Estrogen and the estrogen-receptor antagonist tamoxifen therapy is associated with reduced risk of coronary artery disease events in postmenopausal women. Because of the contribution of inflammation to atherosclerosis, atheroprotective effects of estrogen and tamoxifen may in part be due to favorable changes in vascular responses to inflammatory mediators, as both estrogen and tamoxifen have been shown to have antioxidant properties in lipoprotein oxidation assays. In human coronary smooth muscle cells, tumor necrosis factor (TNF) alpha causes rapid increases in intracellular oxygen species, which are essential for transcription factor (NFkappaB) activation. NFkappaB in turn activates many genes leading to synthesis of proinflammatory proteins. We examined the effect of estrogen or tamoxifen on generation of reactive oxygen species using an intracellular marker (dichlorofluorescein) and confocal laser microscopy for detection. One hour of pretreatment with 17beta-estradiol at physiological concentrations (0.1-10 nM) or tamoxifen at therapeutic concentrations (5-500 nM) dose-dependently inhibited TNF alpha-generated reactive oxygen species and inhibited binding of NFkappaB to DNA (by gel shift assay). Inhibitory effects on reactive oxygen species and NFkappaB binding to DNA were also seen with 17 beta-estradiol, a stereoisomer of estradiol that does not bind to the estrogen receptor. Hydrogen peroxide-generated reactive oxygen species were also inhibited by estrogen and tamoxifen. These early effects of estrogen and tamoxifen appear to require a hydroxyl group in position 3 of the A-ring, since 3-methoxy estrone that lacks this OH-group had no inhibitory effects on reactive oxygen species or on NFkappaB binding to DNA. Thus estrogen and tamoxifen may reduce coronary artery disease risk in part due to anti-inflammatory effects via reactive oxygen species scavenging, resulting in inhibition of proinflammatory gene activation. We found that smooth muscle cells release TNFalpha into the supernatant in response to viral infection and estrogen and tamoxifen attenuate TNFalpha release (by enzyme immuno assay).We demonstrated that TNFalpha transcriptionally activates the major immediate early CMV promoter (MIEP) and estrogen and tamoxifen attenuate this activation which is dependent on reactive oxygen species and NFkappaB. These effects are due, at least in part, to antioxidant properties of 17beta-estradiol, which, unlike 17beta-estradiol, has only very low affinity for the estrogen receptor. We also found that estrogen inhibits TNFalpha-induced COX-2 mRNA, an immediate early inflammatory gene. Finally, pretreatment with estradiol attenuates TNFalpha-induced adhesion molecule-1 (ICAM-1) mRNA expressed in response to oxidant stress. Updated 9/17/1999We further demonstrated in SMC transfected with the COX-2-CAT reporter gene, that exogenous TNFalpha (2-5ng/ml) activates COX-2-CAT (2.4-fold). Pretreatment with hormones/drugs had the following effects: E2 (0.1-1 nM) reduced COX-2 activity by 25% and 50%; 17alphaE2 (0.1-1 nM)inhibited COX-2 by 20 % and 29 %; tamoxifen (50-500 nM= therapeutic doses)reduced COX-2 by 20 % and 46 %, and 3-methoxyestrone (0.1-1 nM) had no effects on TNFalpha-induced COX-2 transcriptional activity, which is dependent on 2 essential NF-kappaB binding sites in the COX-2 promoter. We next demonstrated that exogenous TNFalpha (2-5ng/ml) activates transcription from the major immediate early CMV promoter (MIEP-CAT), and pretreatment with E2 , or with 17alphaE2 (0.1-1 nM each), or with tamoxifen (50-500 nM), but not with 3-methoxyestrone, reduced MIEP-CAT activity by 50 %.TNFalpha is known to generate reactive oxygen species (ROS) and activates NF-kappaB, which in turn activates numerous viral and cellular promoters, among them the MIEP, COX-2, and ICAM-1. Because TNFalpha generates ROS, activates NF-kappaB, and induces ICAM mRNA, we asked whether E2 or 3-methoxyestrone had any effect on ICAM expression. ICAM-1 mRNA was induced at 3hours to 24 hours after exposure of SMC to TNFalpha (2-5ng/ml), and pretreatment with E2, but not with 3-methoxyestrone inhibited ICAM mRNA at 3 hours to 24 hours after TNFalpha.(The ICAM-1 promoter construct we used contains an NF-kappaBsite critical to its activation). In SMC transfected with the an ICAM reporter gene, treatment with TNFalpha resulted in ICAM-1-CAT activity, and pretreatment with E2 (1, 10, 100 nM) dose-dependently inhibited ICAM-CAT. Future studies will address potential estrogen receptor-mediated activity, in addition to the free radical scavenging properties of estrogen.